ABSTRACT Craniofacial sutures are fibrous joints between bones of the skull. Within them, osteoblasts differentiate at the borders of a central suture mesenchyme and are incorporated into growing bone at osteogenic fronts in the highly regulated process of osteogenesis. Skull function requires that sutures: 1) form properly during embryogenesis, 2) allow rapid pre- and postnatal growth, and 3) serve as shock absorbers to dissipate damaging mechanical forces and as reservoirs for stem cells to maintain and repair bone. Mutations in a variety of genes, including signaling pathway components and transcription factors, cause various forms of craniosynostosis (CS). In CS, sutures fuse prematurely, preventing the coordinated growth of the skull and brain. Significant surgical intervention is required to prevent the deleterious neurological consequences of raised intracranial pressure. The wide variation between sutures in terms of form, function, and susceptibility to fusion suggests that gene expression profiles vary considerably among sutures. The currently funded, parent grant ?Transcriptome Atlases of the Craniofacial Sutures? (U01 DE024448-03) addresses this issue. Laser capture microdissection of the separate osteogenic front and suture mesenchyme subregions is being undertaken for up to 11 sutures in wild type mice and in 2 CS mouse models, at different embryonic ages, to create RNA-Seq libraries. However, the expression patterns of genes regulating suture formation and osteogenesis also indicate the presence of cell subpopulations within the osteogenic fronts and suture mesenchyme. Accurate identification of these subpopulations would reveal the patterns of signaling pathway and transcription factor interactions regulating suture patency and osteogenesis. The parent project, by using bulk cell RNA extraction that homogenizes RNA expression within each suture subregion, cannot reveal this organization. This Competitive Revision proposal aims to address this deficit and significantly expand our knowledge of suture biology by providing the means to identify cell subpopulations within the subregions of the wild type murine suture. In this new Specific Aim 4, droplet-based single cell RNA-Seq (scRNA-Seq) will be performed on 4 major cranial sutures (coronal, sagittal, interfrontal and lambdoid), at 3 stages of development (late embryogenesis (E18.5), rapid postnatal growth (P10) and post-weaning maintenance (P28)) to obtain a minimum of 1,000 scRNA-Seq libraries per suture at a given time point. Collections of curated, quality- controlled, scRNA-Seq files will be provided to the FaceBase Hub for use by the wider scientific research community. Analysis of the expression profiles of individual cells will allow the identification of suture cell subpopulations, and elucidate how the various signaling systems, their effectors and readouts are organized and synthesized to regulate suture patency, osteogenesis and other functions at distinct periods of mammalian life. They will provide the basis for new hypotheses of suture formation, maintenance, and function, and provide insight into suture pathologies, with wider significance for bone development in general.